The cytology of the digestive and salivary glands of
the limpet, Patella
ByD. PUGH
(From the Biochemistry Department, Institute of Orthopaedics, Stanmore, Middlesex)
Summary
As reported by earlier investigators, the epithelium of the digestive tubules is composed of two cell-types. One type of cell is glandular, the other type is absorptive and
digestive, and to a lesser extent secretory. The latter type of cell also contains glycogen
and numerous lipid globules, so that the digestive gland as a whole contains a large
quantity of reserve food material. The epithelium of the digestive duct possesses
a single cell-type; the cells are ciliated and heavily pigmented, and they produce a
viscous secretion.
The salivary gland is a compound tubular gland. The cells elaborate a secretion
containing protein and probably some carbohydrate.
Introduction
M A N Y naturalists have included a general description of the limpet in their
studies. The shell and radula have been thoroughly investigated, and several
works have given accounts of the limpet's internal morphology and histology.
The visceral hump of the limpet is brownish green. The pigments responsible for this coloration were thoroughly investigated by MacMunn (1886 a, b;
1889) and Newbigin (1898). MacMunn examined the colour of the pigments
in frozen sections by low-power microscopy, and conducted spectrophotographic analyses of alcoholic or alcoholic ether solutions. In his earlier paper
he concluded that the green pigment was enterochlorophyll, and was elaborated by the animal from plant chlorophyll in the food. He also demonstrated
that the pigment granules did not contain starch or cellulose, and so did not
represent food or symbiotic algae. In a later paper he stated that the pigment
was chlorophyll, although the solution was more stable than a normal solution
of chlorophyll.
Newbigin examined sections of material fixed in formaldehyde solution.
She described the pigment granules of the gut cells and the 'typical molluscan
vesicles' of the digestive gland cells. She examined alcoholic solutions of
digestive glands and faeces and demonstrated enterochlorophyll spectrophotographically. The digestive gland extract turned green on addition of
acid, and the spectrum was altered. Unlike chlorophyll the original spectrum
could be restored by the addition of alkali, and the process repeated several
times. Both investigators recorded that the green solutions of enterochlorophyll displayed a red fluorescence. Newbigin also found a variable amount of
a lipochrome pigment associated with the enterochlorophyll, and MacMunn
remarked on the solubility of enterochlorphyll in lipids.
The salivary glands were first mentioned by Lankester (1867). Griffiths
[Quart. J. micr. Sci., Vol. 104, pt. 1, pp. 23-37, 1963.]
24
Pugh—Digestive glands of Patella
(1888) gave an account of their position and morphology. In 1885 Gibson
wrote the first comprehensive account of the morphology and anatomy of the
limpet. He stated that the cells of the digestive gland tubules were packed
with 'biliary secretion', and mentioned the salivary gland as being a compound
tubular gland. The tubules were lined with cuboid epithelial cells filled with
yellow granules.
Davis and Fleure published a monograph on the limpet in 1903. Then in
1932 Graham published a detailed account of the morphology and histology
of the alimentary canal. He showed that the gut epithelium had a different
appearance in different regions of the gut. The paper included a short description of the salivary gland, a more detailed account of the digestive gland,
and the results of feeding experiments were recorded.
Rees (1934) wrote a paper on the biology of Cecaria patellae, which included a description of the digestive gland of normal and parasitized limpets.
The following account is a report of further investigations on the digestive
and salivary glands of the normal limpet.
Material and methods
Limpets [Patella vulgata) were obtained from the Dove Marine Laboratory,
Cullercoats. They were dispatched by train, in a tin with seaweed, received
the following day, and stored for a week at 40 C. The limpets ingest small
pieces of stone along with fragments of the algae on which they browse, and
this period of starvation helps to eliminate the gritty material from the
animal's gut. The digestive gland closely surrounds the coils of the intestine,
and part of the gut is always sectioned with the digestive gland, so that the
presence of stony material in the lumen of the gut hinders section cutting.
Blocks of digestive gland were cut from the posterior dorsal portion of the
visceral hump. The block included part of the superficial gut coil (fig. 4 in
Davis and Fleure, 1903). The salivary glands were removed whole. In some
types of fixation the entire visceral hump was fixed and dissected afterwards,
because the tissues of the limpet are soft and friable; but when fixatives containing osmium were used, the visceral hump was dissected before fixation.
Of many fixatives investigated it was found that Zenker was the best for the
digestive gland; Susa gave closely similar results. Cytoplasmic inclusions were
well preserved by mixtures containing potassium dichromate, such as Sanfelice's and Regaud's fluids. Flemming-without-acetic was the best fixative for
the salivary gland. This and Zenker were the only fixatives that preserved the
masses of globules visible in fresh preparations. Generally a strongly acid
fixative preserved more of the tissues than a neutral or weakly acid fixative.
Unless otherwise stated, tissue blocks were dehydrated and embedded in
paraffin after fixation. Tissues fixed in formaldehyde solution, with and without postchroming, were embedded in gelatin (Pearse, i960) for the coloration
of lipids and certain other histochemical procedures.
Sections from paraffin blocks were usually cut at 10 /u; 5 /x and 2-5 )u sections
were used for special methods. Formaldehyde material embedded in gelatine
Pugh—Digestive glands of Patella
25
was sectioned at 15 /x. The sections were soaked individually in 30% ethanol
and then floated on water, where they flattened immediately (Holt, 1958).
They were mounted on clean, untreated slides. The sections were mounted
before staining because of the fragility of the material.
Sections from each block were stained as a routine with haematoxylin and
eosin, iron haematoxylin, and toluidine blue. Mitochondria were demonstrated by the Kull and Altmann stains. Methyl green / pyronin staining,
and Feulgen and plasmal reactions, were used on suitably fixed sections.
Various histochemical methods were used in an attempt to ascertain the
nature of the cells' contents, particularly the massed granules of one type of
digestive gland cell, here called cell type A. Sections were stained by the
periodic acid / Schiff method (PAS), buffered methylene blue, and mucicarmine to demonstrate compounds containing carbohydrates.
Lipids were stained with Sudan black, Sudan III and IV, Nile blue, oil red,
and Baker's acid haematein. The tetrazonium and Millon's reactions were
used to stain protein. Frozen sections were examined for /?-glucosaminidase
and jS-glucuronidase, with AS-LC naphthyl glycosides in a simultaneouscoupling azo-dye technique (Pugh and Walker, i960).
Fresh material was examined in teased preparations and squashes.
Observations
The digestive gland
As stated by several authors (Gibson, 1885; Davies and Fleure, 1903;
Graham, 1932), the digestive gland occupies the greater part of the visceral
hump, closely investing the coils of the intestine. The gland is composed of
a mass of tubules, which are not arranged in definite lobes. The tubules are
lined by a single layer of cells surrounded by a thin layer of connective tissue.
The epithelial cells of the tubules are of two types, here designated cell types
A and B. Cell type A is more numerous than cell type B; the latter cells frequently occur in groups of two or three. The ducts of the digestive gland
empty into two main trunks which fuse to give a single duct opening into the
stomach. The columnar epithelium lining the ducts is composed of a single
cell-type.
Fresh preparations. Small pieces of digestive gland were examined fresh,
teased in saline or in the fluid lying between the shell and the mantle of the
limpet. Cell type A is full of globules, usually lightly tinted green; some may
appear colourless, but the majority are coloured. A large apical granule is
brownish green and appears to consist of a mass of smaller granules. Newbigin
(1898) found than an alcoholic solution of digestive gland gave a red fluorescence in ultraviolet light. The globules of cell type A examined fresh with
ultraviolet light show a bright red fluorescence; the apical granule appears
dark red, almost brown. The cells are often disrupted, and rounded cell
fragments consisting of either a nucleus or apical granule and a fragment of
cytoplasm, occur frequently. Whole cells detached from the basement membrane have a tendency to assume a spherical form, and after being removed
26
Pugh—Digestive glands of Patella
from the animal for several minutes, any remaining intact cells will have
assumed this shape. By phase-contrast microscopy the globules of cell type A
appear refractile and the nucleus may be observed. The apical granule can be
seen to consist of an aggregate of small granules in each of which the pigment
is concentrated at the periphery. Granules which are possibly mitochondria
can be seen either in or on the apical granule, and in the cytoplasm of the cell
fragments. Cilia have not been observed in these gland cells. Cell type B
does not contain any of the globules which fill the type A cells. By phasecontrast microscopy the cell may be seen to be packed full of mitochondria,
and the nucleus appears as a large refractile body. The cell has a tendency
to round up, but does not fragment as easily as does cell type A. The cells of
the digestive ducts are ciliated. Each cell possesses four cilia, two of which are
longer than the other pair. These cells have the upper half of the cell filled
with bright green pigmented granules.
Stained preparations: Cell type A. Cells of type A are narrow columnar
cells from 18 to 30 [j. in height and from 4 to 5 ju. in breadth, in fixed preparations. The lateral membranes of the cells are well preserved after Zenker
fixation, but difficult to distinguish after some types of fixations, such as
formaldehyde. The tips of the cells may be flattened, rounded, or apparently
in process of being separated from the cell. In the cells with flattened tips
the apical layer of cytoplasm is finely granular, and often appears vertically
striated; this layer of cytoplasm is denser than that of the remainder of the
cell. Distended cell tips show a vacuolated region with an outer rim of dense
cytoplasm (fig. 1). All gradations between these two extremes occur.
Immediately below this layer, in all cells of this type, there lies a brownishgreen Iobed granule (which will be referred to as the apical granule) within
a vacuole. This granule is 3 to 4 /u, in diameter. It is removed by fixation in
alcohol, or by fixation in formalin and subsequent dehydration. It is well
preserved by fixatives containing dichromate.
The greater part of the cell is filled with granules of varying size. After
Zenker fixation followed by haematoxylin and eosin staining, the granules
stain pink; usually the largest granules are the palest and the staining intensity
increases with reduction in granule size. Comparison with a formaldehydefixed gelatin-embedded section, or a Flemming-without-acetic preparation,
shows that there are in addition small granules containing lipid among the
other granules. These are particularly numerous at the base of the majority
of the cells and they occur throughout the cytoplasm of a few cells. Mitochondria may be stained by iron haematoxylin, Altmann, or Kull stains after
Zenker or osmium fixation. They appear as small ovoid bodies scattered
throughout the cytoplasm between and often clustered around the granules,
and a few lie over the apical granule.
The nucleus is situated near the base of the cell. It is 2-5 to 3 ju, in diameter,
and contains a central nucleolus and some scattered chromatin, both of which
are Feulgen-positive. The nucleus stains purple with methyl green/pyronin.
After Mallory's triple stain the apical cytoplasm stains red, while the remain-
Pugh—Digestive glands of Patella
27
der of the cytoplasm and the apical granule are unstained. The mitochondria
stain red, and the cytoplasmic granules blue.
Cell type B. Cells of type B are fewer in number than those of type A.
They are triangular. The cell base varies from 7 to 10 /x and the cell tapers to
a narrow neck where it reaches the lumen. The cells are from 15 to 20 ^i in
height.
secretion
droplet
apical granule
cell type A
cell type 8
protein
granule
retractile
lipid
sphere
globule
mitochondria
— nucleus
nucleus
FIG. I. Cells of digestive gland; composite drawing.
In fixed and stained preparations the cytoplasm is foamy and basiphil.
After formaldehyde fixation the cytoplasm stains homogeneously, but after
Zenker, Susa, or osmium fixation, refractile spheres may be distinguished
(fig. i). These spheres stain an intense red with Altmann's acid fuchsin, and
Kull's triple method; by the latter method the surrounding cytoplasm stains
blue.
The mitochondria of cell type B are more numerous than those of cell
type A. They are scattered thickly throughout the cytoplasm. Because of the
intensely basiphil staining of the cytoplasm the mitochondria are difficult to
distinguish after iron haematoxylin, but can be demonstrated by Altmann's
method, and by Sudan black staining of material fixed in Zenker and embedded
in paraffin.
The nucleus (4 /x in diameter) is larger than that of cell type A, and has a
central nucleolus 1 to 1-5 /u in diameter. The nucleolus, nuclear membrane,
and chromatin granules are strongly Feulgen-positive; the nuclear sap and the
basiphil cytoplasm are weakly positive. Staining with methyl green / pyronin
colours the nucleus purple. The cytoplasm stains purple except for the cell
tip, which has a reddish, vacuolated appearance; the refractile spheres stain in
shades of blue, grey, or pink. After Mallory's triple stain the mitochondria
stain red, the cytoplasm deep orange, and the refractile spheres pale orange.
28
Pugh—Digestive glands of Patella
Digestive duct cells. The digestive ducts are lined by narrow, ciliated,
columnar epithelial cells. The walls of the two main ducts are slightly ridged.
The epithelium is composed of a single layer of cells; the increased width
of the ridges is caused by the greater length of some individual cells. These
cells are 3 \x in width and range from 16 to 35 p in height. The cells of the
smaller ducts are of uniform size,
measuring 3/A in width and 21 /n in
secret/on droplet
height, with only slight variations.
Apart from the variations in height,
all the duct cells are essentially similar
(fig- 2).
pigment granule
The cilia may be observed in fresh
preparations. They are rarely preserved in fixed preparations, but the
material lying outside the cell tips has
a striated appearance as if its position
mitochondrion
was determined by ciliary action. This
extracellular material consists of debris
and of clear globules, some of which
appear to be in process of being extruded from the epithelium (fig. 2).
The apical layer of cytoplasm is
granular and stains deeply with eosin.
Below this is a layer of small pigment
Epithelium of digestive duct; comgranules, which usually fill about half
posite drawing.
of the cell. These granules appear
bright green when examined fresh, and olive green after fixation.
The nucleus lies below the pigment granules in the basal third of the cell.
The cells of the main duct have oval nuclei measuring 3 to 3 -5 /n, their longer
diameter lying parallel with the long axes of the cells. Transversely, they
occupy the width of the cell. The nuclei of the small duct cells are round,
3 fj, in diameter, and also take up the entire cell width. All the nuclei have a
small central nucleolus, clear sap, and several chromatin granules. The nuclei
are Feulgen-positive. The mitochondria are small and scattered throughout
the cell. They are rather more numerous in the pigment layer and lie close to
the lateral cell membranes. The nucleus stains purple with methyl green /
pyronin. After Mallory's triple stain the apical cytoplasm and mitochondria
stain red, the nucleus blue, and the basal cytoplasm orange.
Lipid. Lipid has been detected by examining unstained osmium-fixed
sections and formalin-fixed, gelatin-embedded material. 10% aqueous formaldehyde does not preserve the digestive gland cells very well, so the effect of the
addition of buffers or salts, with and without postchroming, was tried (table 1
in appendix, p. 37). Postchroming material fixed with formaldehyde calcium,
or adding sea-water to this solution improves the fixation, without altering the
staining properties of the cells.
Pugh—Digestive glands of Patella
29
In frozen sections the small lipid droplets of cell type A colour with Sudan
black in 70% ethanol. These droplets were removed during the dehydration
of blocks fixed in any fluid which did not contain osmium. Sudan black
colouring of Zenker-fixed paraffin sections coloured black only the mitochondria and small granules lying on the apical granule (of similar size to
mitochondria). Sections fixed by Flemming-without-acetic were treated as
described by Wigglesworth (1957); the mitochondria and smaller fat granules
could then be seen more distinctly. Cell type B does not contain any fat
granules; the cell is unstained in frozen sections treated with Sudan black. In
formalin-fixed frozen sections and Zenker-fixed paraffin sections the pigment
granules of the digestive duct cells were coloured black with an alcoholic
solution of Sudan black. These granules were also blackened by osmium.
The picture obtained by colouring with Sudan III, Sudan IV, and oil red
(Pearse, i960) is similar to that obtained with Sudan black. Several methods
(Cain, 1950) have been used in an attempt to discover the nature of the lipids
(table 2). From the results obtained it seems possible that the fat granules of
cell type A contain a simple lipid which may be unsaturated and that the
apical granule contains enterochlorophyll. The granule of the duct cells
appear to contain enterochlorophyll and a lipid, which is probably a mixture
of phospholipid and lipochrome.
Compounds containing carbohydrate. Sections protected by collodion were
treated by the PAS method, either without prior treatment or after digestion
with either diastase (saliva) or hyaluronidase (Benger Ltd., Holmes Chapel,
Cheshire). In untreated sections the granules and cytoplasm of cell type A
stained intensely, so that the granules were indistinguishable at low magnifications. Cell type B stained moderately. The apical cytoplasm of the duct
cells stained deeply and the remaining cytoplasm lightly; the pigment
granules were unstained.
Treatment with diastase reduced the amount of colour developed in the
cytoplasm of cell type A with PAS. The granules were unaffected by diastase.
A reduction of PAS-positive material occurred in the cytoplasm of cell type B.
Hyaluronidase digestion removed a greater quantity of PAS-positive material
than did treatment with diastase. In some parts of a tubule hyaluronidaselabile material was removed from the whole of cell type A; in other parts
from the distal region only. Material was removed from both granules and
cytoplasm. Cell type B stained rather less intensely after hyaluronidase treatment than cells of this type in a control section. Treatment with diastase or
hyaluronidase slightly reduced the amount of colour developed with PAS in
the duct cells. These results suggested the presence of glycogen and a
mucoid substance; further tests were made for both compounds.
Mucoid substances. As reported by Graham, it was found that the granules
of cell type A give a negative reaction with mucicarmine. The cell type A
granules stained lightly and the connective tissue deeply with alcian blue.
No other part of the digestive gland stained with this dye.
Toluidine blue stained the granules of cell type A red purple, and the
30
Pugh—Digestive glands of Patella
whole of cell type B deep purple in aqueous mounts. This metachromasia
was partially removed by dehydration. The apical cytoplasm of the duct cell
appeared pink and the remainder of the cell purple in aqueous mounts; after
dehydration the former appeared purple and the latter blue.
A methylene blue extinction curve was obtained from Zenker-fixed
sections at pH 2-4 to 8-o (Pearse, i960). At pH 5-2 and higher the digestive
tubules stain an intense blue-black. Below this pH the staining becomes less
intense. At pH 4-0 the cytoplasm of cell type A fails to bind methylene blue
and the granules bind the dye weakly. Cell type B binds methylene blue
strongly at pH 4-0 and moderately at pH 2-4. At pH 3-6 and below the apical
cytoplasm of the duct cells was unstained; the basal cytoplasm stained
lightly at pH 2-4. The cells stained deeply at pH 6-o and above. The pigment
granules failed to bind methylene blue at any pH value.
A water homogenate of digestive gland tested for hexuronic acid by the
carbazole method (Dische and Borenfreund, 1951) gave a negative result.
A similar homogenate examined chromatographically (Leaback and Walker,
1957) gave a positive reaction for hexosamine.
Glycogen. Attempts to confirm the presence of glycogen by Best's carmine
method have been inconclusive. The cytoplasm of cell type B stained deeply
with the carmine solution and also with the haematoxylin counterstain; the
base of cell type A stained lightly. Iodine stained material in both these sites,
but not in the digestive duct cells.
Glycosidases. Cell type B gave a negative result for /J-glucosaminidase,
while cell type A showed a high activity. The cytoplasm between the granules
was deeply, and the apical region of the cell very deeply stained. The duct
cells stained lightly and diffusely. Cell type A showed a very high /S-glucuronidase activity. There was some variation between cells, but usually the dye
was deposited throughout the cytoplasm, increasing in quantity towards the
tip, which stained intensely. Cell type B stained deeply and diffusely. The
apical cytoplasm of the duct cells was deeply, and the remainder of the cytoplasm lightly stained. The pigment granules were unstained. The addition
of the principal salts of sea-water to the incubation mixture markedly increased
the /S-glucosaminidase activity in sections. Homogenates of digestive gland
assayed for this enzyme in the presence of artificial sea-water (Sverdrup,
Johnson, and Flemming, 1942) showed an enzyme activity 5 to 10 times that
of the control. Additions of sodium chloride to mammalian tissue homogenates have been shown to cause a slight increase in enzyme activity (Pugh,
Leaback, and Walker 1957). Such additions of salt have no obvious effect on
the activity of j8-glucuronidase.
Protein. In sections stained by the tetrazonium method the nucleus, cell
membrane, and cytoplasm were moderately stained, and the apical granule
and cytoplasm deeply stained. Most of the cytoplasmic granules stained
deeply, the larger granules stained more heavily than the small granules
(lipid globules were not preserved in these preparations). The cytoplasm
and nucleus of cell type B stained deeply; the refractile spheres were
Pugh—Digestive glands of Patella
31
moderately stained. Sections treated with Millon's reagent stained similarly,
but less intensely than those stained by the tetrazonium method.
The salivary glands
The limpet possesses four orange-coloured salivary glands, which lie at the
front of the visceral hump. There is a dorsal and ventral gland lying on each
side of the fore-gut. The salivary gland is a difficult tissue to study histologically because very few fixatives preserve all of its structure, and the appearance
of fixed material varies greatly according to the fixative used. As stated
earlier, of the fixatives tried, only Zenker and Flemming-without-acetic preserved all of the material observed in fresh preparations.
The salivary glands are of similar structure: the only differences observed
between them are consistent with the individual glands being in a different
functional state, and the following account applies to all the salivary gland
tissue. The salivary gland is a compound tubular gland. The tubules are
surrounded by a thin layer of connective tissue, and are arranged in lobes
surrounded by a thicker layer of connective tissue. The lumen of individual
tubules is often difficult to distinguish. The salivary ducts are lined by cuboid
epithelium; the cells possess large central nuclei.
After fixation in Flemming-without-acetic followed by staining with haematoxylin and eosin, the cells show great variation in their contents and staining
properties. Some cells appear pale yellow with finely granular cytoplasm,
others contain small globules which stain pink. The remainder of the salivary
gland cells contain globules which range in size from these small ones to
large globules, one of which may fill an entire cell. Occasionally one of
these large globules may be seen to be in process of being extruded from the
cell. The globules stain increasingly basiphil with increase in size. The
largest globules stain purple with the haematoxylin.
A single tubule usually contains cells showing all variations, i.e. those
possessing granular cytoplasm, and cells with all sizes of globules. Individual
cells appear to contain only one size of globule. The amount of cytoplasm
visible in a cell varies inversely with the size of the globule that it contains.
For convenience the cells which appear granular are referred to as type 1 cells;
the others which contain graded sizes of globules are grouped together as
type 2 cells (fig. 3).
After Flemming-without-acetic or Zenker fixation, type 2 cells measure
from 7 to 13 fj, at the base, the majority approximately 11 /x, and have a height
of 10 to 13 fi with an average of 11 to 12 (x\ the cells taper slightly towards the
centre of the tubule. Type 1 cells are more angular in appearance, the lengths
between two corners varying from 7 to 13 /x with an average of 9 ju..
Type 2 cells have peripheral nuclei which are small and ovoid, with their
greater diameter about 3 /x. They appear granular, but do not have nucleoli.
The nuclei are Feulgen-positive. The small quantity of cytoplasm usually
present in these cells stained lightly with plasma dyes.
The nuclei of type 1 cells were either peripheral, eccentric, or central in
32
Pugh—Digestive glands of Patella
position. They were spherical with a diameter of 3 JX, and resembled the
nuclei of type 2 cells by having a granular appearance, and being Feulgenpositive.
After the majority of fixatives the differences between the two types of
salivary gland cells were markedly increased. Cell type 2 was empty except
for a nucleus or contained a small quantity of cytoplasm and possibly a few
granule
type 1 cell
nucleus
bosiphil cytoplasm
FIG. 3. Cells of salivary gland; composite drawing.
small globules. The cell had a heavily marked outline and a peripheral
nucleus with its long axis lying parallel with the cell membrane. Type 1 cells
stained more deeply basiphil than did the cell type B of the digestive gland,
and no nuclei or cytoplasmic structure could be observed.
Altmann and Kull stains after Regaud, Schridde, Zenker, or Flemmingwithout-acetic fixations showed the mitochondria to be tiny spherical bodies
in both cell types. The largest globules stained intensely red with Altmann's
acid fuchsin. If Kull's triple stain was used, the globules stained in shades of
red, orange, and blue, the largest red and the smallest blue. Type 1 cells
appeared solidly red after Altmann's stain; if highly differentiated the cell
could be seen to contain mitochondria. If Kull's method was used the cytoplasm stained deep blue or green, and the mitochondria red.
In sections stained by Mallory's triple stain the cytoplasm of type 1 cells
stained blue, and some cytoplasmic granules deeper blue. The large globules
of cell type 2 stained red, the small globules and cytoplasm orange, and the
nuclei of both cell types blue.
The nuclei of all the salivary gland cells stain green or purple with methyl
green / pyronin. After formaldehyde fixation the cytoplasm of cell type 2
stained pale pink by this method. In material fixed in Zenker the cell type 2
globules also stained pink; most of this colour could be removed by ribonuclease digestion. The cytoplasm of cell type 1 stained intensely pink in
Pugh—Digestive glands of Patella
33
sections fixed by either method; deeply stained granules occurred in the
cytoplasm. In sections treated with ribonuclease the type 1 cell appeared
larger than in the control and the cytoplasm stained palely. The granules
were larger and globular in appearance; they were similar in size to the small
acidophil globules of some 2 cells.
Lipid, After formaldehyde fixation and postchroming the cytoplasm of type
1 cells coloured moderately, and scattered cytoplasmic granules deeply, with
Sudan black. The same result was obtained with material fixed in Zenker and
embedded in paraffin. These cells were not blackened by osmium. The other
staining reactions of the type 1 cell lipid, which are summarized in table 2,
suggest that it is phospholipid.
Substances containing carbohydrate. Sections were stained by the PAS
method, either untreated or after enzyme digestion as for the digestive gland
sections. In untreated sections the cytoplasm and granules of type 1 cells
stained intensely. The outlines of type 2 cells stained lightly; the globules
when present were unstained. Sections treated with hyaluronidase did not
show any difference in staining properties from the untreated sections. Prior
incubation with diastase did not affect the staining of type 2 cells. The
granules of type 1 cells stained as intensely as in the control, but the surrounding cytoplasm stained lightly; so a considerable quantity of PASpositive material had been removed by digestion with diastase.
Glycogen. Type 1 cells were intensely basiphil, and the same difficulty of
staining with Best's carmine stain for glycogen was encountered as with the
digestive gland, and the results were inconclusive. The presence of glycogen
was confirmed by iodine staining in cell type 1.
Mucoid substances. In sections stained with toluidine blue and examined
wet, type 1 cells stained purple and the cytoplasm of type 2 cells lighter purple
or blue. Mounting in balsam did not destroy the metachromasia or type 1
cells, but reduced to some extent that of type 2 cells. In sections mounted
in balsam, granules were visible in type 1 cells which stained more deeply than
the surrounding cytoplasm. The type 2 cell globules did not stain, except for
an occasional faint purple coloration. An attempt was made to stain these
globules by using different concentrations of toluidine blue and by the addition of wetting agents.
Increasing the dye concentration above 0-5% did not cause any part of the
gland to stain more intensely. The addition of wetting agents (triton X-100
and span 20, from Charles Lennig, London, W.C. 1) at concentrations of
o-i to 10% caused the globules of type 2 cells to stain purplish pink. Cell
type 1 stained intensely and uniformly purple.
A methylene blue extinction curve was performed on salivary gland
sections. Type 1 cells bound methylene blue strongly at pH 4-0 and moderately at pH 2-4. The cytoplasm of type 2 cells stained palely at pH 4-0 and
did not stain at pH 2-4. The globules stained at pH 6-o and higher pH; they
were unstained at more acid pH.
Glycosidases. Neither ^S-glucosaminidase or /?-glucuronidase have been
34
Pugh—Digestive glands of Patella
demonstrated in the salivary glands. Tissue homogenates and histochemical
methods were used.
Protein. In sections stained by the tetrazonium method, type i cells stained
moderately after Zenker fixation. After formaldehyde fixation type i cells
stained more deeply and the granules could be distinguished as intensely
stained dots. Type 2 cells stained lightly after both types of fixation. The
globules, which were only preserved by Zenker fixation, also stained, the
larger deeply and the smaller globules moderately. Millon's reagent left
the cytoplasm and globules of type 2 cells unstained. Type 1 cells stained
orange pink. Granules were visible but did not differ in colour from the surrounding cytoplasm.
Discussion
Digestive gland
The staining reactions of cell type A suggest that this cell may have several
functions. The presence of glycogen and a large quantity of fat suggests that
the digestive gland may function as a food depot. Barry and Munday (1959)
have reported glycogen storage in limpet tissues. For the visceral hump they
give a mean value of 2% by weight from July to November, decreasing during
November to January to a value of 0-3%, which persisted until March when
the level rose again. The limpets examined have been killed at intervals during
the year, and there has been no obvious difference in the amount of glycogen
found in individual cells. But there was considerable difference in the size
of the digestive gland throughout the year. It decreased in size as the gonad
increased, then grew again in size after the gonad had shed its contents.
This supports the suggestion that the digestive gland functions as a food depot.
The apical granule is similar in form in cells of type A, and contains a derivative of chlorophyll named enterochlorophyll by MacMunn. The remaining
cytoplasmic granules may represent absorbed material with mucus from the
gut, or droplets of secretion elaborated by the cells to be discharged into the
lumen. The staining reactions of these granules suggest that they contain a
substance of high molecular weight which has some of the properties of an
epithelial mucin, and a small quantity of enterochlorophyll. The appearance
of the tips of the cells and the rinding of isolated pieces of cytoplasm in the
tubule lumen suggest either excretion or secretion by the cells. The results
obtained from preliminary feeding experiments (unpublished results) supports the latter suggestion.
/3-Glucuronidase is associated with the degradation of mucosubstances
in mammalian tissues (Walker, i960). It is possible that this enzyme may act
on ingested mucus. This enzyme has been reported to have a possible digestive function in limpets (Corner, Leon, and Bulbrook, i960) and in Helix
pomatia (Billet and McGee-Russell, 1955). The possibility of the enzyme
having a digestive function is the more likely as it occurs in both types of
digestive gland cell; however, the enzyme may possess both functions.
j3-Glucosaminidase occurs only in cell type A; this enzyme is stated to be
Pugh—Digestive glands of Patella
35
associated with mucosubstances, but as far as is known it has not been supposed to have a digestive function in herbiverous molluscs.
Unlike cell type A, type B cells contain only a small quantity of glycogen
and no fat granules. The cell contains numerous mitochondria and has a
high protein content, both of which are characteristic of glandular cells.
The cell also contains a considerable quantity of ribonucleic acid. The refractile spheres contain lipoprotein and possibly DNA. They are probably secretion droplets that will be released into the tubule lumen. During the feeding
experiments referred to above, it was found that these spheres increased in
number several hours after the limpet had been fed. The results obtained
during this investigation of the digestive gland are fundamentally in agreement with those of Graham (1932).
The cells of the digestive ducts are of a single type. Each cell is ciliated and
also has a secretory function. The distal part of the duct cell is filled by
numerous pigment granules which contain enterochlorophyll and phospholipid. In addition the granules contain a lipid which is probably lipochrome.
This pigment was stated to occur in alcoholic extracts of digestive gland
(Newbigin, 1898). It is unlikely to be derived from the digestive gland cells,
which contain a simple lipid only. The apical cytoplasm contains glycogen,
a hyaluronidase-labile material, and much protein; these substances are
found in the scanty basal cytoplasm in smaller quantities. The cell secretes
into the duct lumen a viscous material which is presumably elaborated by the
apical cytoplasm. This secretion behaves as an epithelial mucin assisting
the transport of food material along the duct. The secretion is viscous and
differs markedly from mammalian mucin in its staining properties.
Salivary glandIt has been stated (Graham, 1932) that the salivary gland produces a
lubricating fluid which is poured on to the radula as the limpet feeds. It seems
possible that all or most of the salivary gland cells are engaged in the elaboration of a viscous secretion which contains protein, no lipid, and little or no
carbohydrate. However, the cytoplasm of cell type 1 contains a small quantity of glycogen. The globules of cell type 2 do not show the staining reactions
of a mucosubstance with any method used. The globules may possess a
membrane which is impervious to water, as they were unstained by an
aqueous solution of toluidine blue but coloured by a solution containing a
wetting agent.
The cytoplasm of cell type 1 contains protein, ribonucleic acid, and some
glycogen and lipid. After ribonuclease digestion small replacement globules
appear in place of the granules seen in control sections. At high magnifications the original granules may sometimes be seen lying at the periphery of the
globules or between them, and this suggests a close association between the
two bodies. The whole cell increases in size after ribonuclease digestion in
addition to globule formation. The globules visible after ribonuclease digestion are similar in appearance to the small eosinophil globules of some type 2
36
Pugh—Digestive glands of Patella
cells. This supports the suggestion that type 1 and type 2 cells represent two
stages in a secretory cycle and do not differ greatly in function. The final
stage of development of the cycle would be the cell containing a single large
globule with only a narrow lining of cytoplasm and a peripherally displaced
nucleus. After extrusion of the globule the cell may regenerate and re-enter
the cycle as a type 1 cell, or it may be replaced.
The author wishes to thank Dr. S. M. McGee-Russell of the Virus Research
Group, Medical Research Council, and Dr. P. G. Walker, Head of the
Biochemistry Department, for help and encouragement during the preparation of this paper. This work forms part of a thesis accepted for the degree of
M.Sc. by the University of London.
References
BARRY, R. J. C, and MUNDAY, F. A., 1959. J. mar. biol. Assoc. U.K. 38, 81.
BILLET, F., and MCGEE-RUSSELL, S. M., 1955. Quart. J. micr. Sci., 96, 35.
CAIN, A. J., 195°- Biol. Rev., 25, 75.
CORNER, E. D. S., LEON, Y. A., and BULBROOK, R. D., i960. J. mar. biol. Assoc. U.K. 39, 51.
DAVIS, J. A., and FLEURE, H. J., 1903. Patella (L.M.B.C. Memoir). London (Williams and
Norgate).
DISCHE, Z., and BORENFREUND, E., 1951. J. biol. Chem., 92, 583.
GIBSON, R. J. H., 1885. Trans, roy. Soc. Edinb., 32, 601.
GRAHAM, A., 1032. Ibid., 57, 287.
GRIFFITHS, A. B., 1888. Proc. roy. Soc, 44, 325.
HOLT, S. J., 1958. General cytochemical methods, x, 375- New York (Academic Press).
LANKESTER, E. R., 1867. Ann. Mag. nat. Hist., 20, 334.
LEABACK, D. H., and WALKER, P. G., 1957. Biochem. J., 67, 22P.
LEE, B., 1950. The microtomist's vade-mecum, n t h ed. London (Churchill).
MACMUNN, C. A., 1886a. Phil. Trans, roy. Soc, 177, 235.
18866. Ibid., 177, 267.
1899. Proc. roy. Soc. B, 64, 436.
NEWBIGIN, M., 1898. Quart. J. micr. Sci., 41, 391.
ORTON, J. H., SOUTHWARD, A. J., and DODDS, J. M., 1956. J. mar. biol. Assoc. U.K., 35, 149.
PEARSE, A. G. E., 1960. Histochemistry. 2nd ed. London (Churchill).
PUGH, D., LEABACK, D. H., and WALKER, P. G., 1957. Biochem. J., 65, 16P.
and WALKER, P. G., i960. J. Histochem. Cytochem., 9, 103.
REES, F. G., 1934. Proc. zool. Soc. Lond., 45, 45.
SVERDRUP, H. V., JOHNSON, M. W., and FLEMMINC, R. H., 1942. The oceans. New York
(Prentice Hall).
WALKER, P. G., i960. Biochem. J., 75, 4P.
WIGGLESWORTH, V. B., 1957. Proc. roy. Soc. B, 147, 185.
Pugh—Digestive glands of Patella
37
Appendix
TABLE I
Preservation of the granules of digestive gland cell type A
Fixative
Additions
10% formalin
(Baker's fixative)
10% formalin
(Baker's fixative) followed
by postchroming
10% formalin* in sea-water
solution
10% formalin* in sea-water
solution
Lipid globules
Cytoplasmic
granules
O I
M CaCl2, pH 70
O
0
o-i
M CaCl2, pH 7-0
+
O
O I
M (CH3COO)2Ca, pH 5-5
0 1
M CaCl2, pH 70
±
+
O
4-
+ , preserved. ± , lipid globules preserved but disrupted. O, not preserved. • A solution
containing the principal salts of sea-water was used to give a final solution of salinity about
3-55%TABLE 2
Digestive gland
cell type A
Digestive duct
cell. Pigment
granules
Salivary gland
type 1 cell
Method
Lipid globules
Apical granule
Baker's acid haematein
Alcoholic sulphuric acid
O
pale green
O
bright green
Schultz test without
mordanting
Schultz test with mordanting
Roe-Rice pentose reaction
Molisch reaction
Nile blue, 1 % .
Nile blue, O'Z%
Plasmal reaction.
0
O
0
O
tr
O
0
0
O
O
O
0
0
blue or pink
blue or pink
+
O
blue green
green*
O
O
blue or purple
blue or green*
O
blue
blue
O
0
blue
blue
Cytoplasm
Granules
O
+
moderately bright pale green pale green
green
O
O
tr
+
tr
+
O, no reaction. + , positive reaction, tr, weakly positive reaction. * natural colour of granule.